1Vestibular Neurogenetics Laboratory, Boys Town National Research Hospital, Omaha, Nebraska, United States of America.

Abstract

Otoconia are bio-crystals anchored to the macular sensory epithelium of the utricle and saccule in the inner ear for motion sensing and bodily balance. Otoconia dislocation, degeneration and ectopic calcification can have detrimental effects on balance and vertigo/dizziness, yet the mechanism underlying otoconia formation is not fully understood. In this study, we show that selected matrix components are recruited to form the crystal matrix and sequester Ca(2+) for spatial specific formation of otoconia. Specifically, otoconin-90 (Oc90) binds otolin through both domains (TH and C1q) of otolin, but full-length otolin shows the strongest interaction. These proteins have much higher expression levels in the utricle and saccule than other inner ear epithelial tissues in mice. In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the null mice leads to drastically reduced matrix-Ca(2+). In vitro, either Oc90 or otolin can increase the propensity of extracellular matrix to calcify in cell culture, and co-expression has a synergistic effect on calcification. Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins. Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

Quantitative real-time RT-PCR (qRT-PCR) showed that both Oc90 and otolin had significantly higher expression levels in the utricle and saccule than the ampulla, canal or cochlea in wt mice. The mRNA levels were normalized to that of β-actin (relative mRNA level), and then different tissues were compared to the utricle and saccule whose relative mRNA level was set to 1 (A, B). In (C, D), the relative mRNA level in the E17.5 cochlea was set to 1. The vestibular samples in C and D mainly consisted of the utricle+saccule with a small part of the ampulla. ** and *** denote p<0.01 and 0.001, respectively, when the utricle+saccule are compared with other tissues (n = 3 for each type of tissue in each age group). ### and $$$ denote p<0.001 when P7 is compared with E17.5 and P0, respectively.

Inorganic calcium deposits were visualized with Alizarin Red S staining, and averaged percentages of cells with ECM calcification over total cells were compared for different constructs and vectors. Under the same inducing conditions, untransfected cells and cells transfected with empty vectors (A) had similar ratios of calcification nodules, but both had significantly lower ratios than those transfected with otolin (B), Oc90 (C), or Oc90+otolin (D) (Figure 5B, 5C or 5D vs. 5A, p<0.001 as denoted by ***, n = 3 experiments x 3 fields for each). Figure 5E shows histograms of the averaged percentages of cells with ECM calcification. Co-transfection of Oc90 and otolin had a synergistic effect on calcification. # and ### indicate p<0.05 and 0.001, respectively, when pOc90 or pOc90+pOtolin is compared with pOtolin. $ indicates p<0.05 when pOc90+pOtolin is compared with pOc90.

The collagen-like domain, aka triple helix (TH) domain, of otolin and Col10a1 have a repeating (Gly-X-Y)n (the shaded ‘G’s denote Glycine), where X is frequently Proline and Y hydroxyproline. The TH domain starts at residue 130 and ends at residue 342 in otolin. The C-terminal globular C1q (gC1q) domain starts at residue 343 in otolin. In collagen, this domain is also known as the non-collagenous NC1 domain. The two D's (blue) denote aspartic acids in Col10a1 that provide the ligands for a cluster of four Ca2+ ions. These residues are conserved in otolin. The inter-subunit contacts are almost entirely hydrophobic with key contact residues highlighted in yellow. Most of these residues are conserved in otolin. Residues 1–23 in otolin and 1–18 in Col10a1 are the signal peptides, respectively; residues 19–56 in Col10a1 is the non-collagenous NC2 domain, and 24–36 in otolin is the residual NC2-like domain. * : . indicate identical residues, conservative and semi-conservative variations, respectively, between otolin and Col10a1.

(A) The tertiary structures of Col10a1-C1q domain (green) and otolin-C1q domain (magenta) are nearly super-imposable. The modeled structure is consistent with the crystal structure of human COL10A1. Ca2+-binding residues are labeled and projected as sticks. (B) The surface of the otolin-C1q domain with temperature factors denoted in different colors. (C) Clustering of residues and the cationic surface of otolin-C1q domain. Residues are colored according to their electrical charges and hydrophobic nature. The nine key cationic residues are labeled and projected as sticks. (D) The protein backbone RMSD during 1ns simulation. After 400ps, the structure is stable. The 400–1000 ps range was used for computing the final structure. (E) The temperature factor (RMS fluctuation) of each residue. The residues with higher RMS fluctuation usually are exposed on molecule surface and may construct active sites. The Ca2+-binding residues in (A) are labeled.